Quantitative enzyme histochemical methods have been applied to determine kinetic parameters of enzymes in intact unfixed tissue sections to obtain information on behaviour of enzymes in their own cellular environment and zonal differences in their function within a tissue (see references 1A-6A) (References are immediately above). These studies demonstrated that both variations and regional differences in the kinetic parameters of several enzymes occur which partly explain the enormous plasticity of tissues to adapt to alterations in the environment. Although unfixed cryostat sections is one step closer to the in vivo situation than homogenates, they still do not provide information on how enzymes behave in vivo. We want to establish the exact role of proteases in physiological and pathophysiological processes. Examples are turnover of collagen (7A-9A), activation of the immune system (10A), arthritis (11A,12A) and metastasis of cancer (13A,14A). For such studies, methods to measure protease reactions in individual living cells are needed. To visualize protease activity in single cells, a new class of fluorogenic substrates for proteases containing cresyl violet was synthesized. This is a highly fluorescent leaving group after proteolytic cleavage of the amide bonds.
Dipeptidyl peptidase IV (DPPIV) is an ectopeptidase present at the plasma membrane of many cell types. It is a transmembrane glycoprotein with a short cytoplasmic tail, one hydrophobic transmembrane segment and a large extracellular domain (15A). DPPIV is involved in activation of bioactive molecules such as cytokines (16A,17A) and it participates in the extracellular digestion of polypeptides to provide substrates for peptide and amino acid reabsorption (18A,19A). In hepatocytes, the enzyme is present at the apical bile canalicular membrane and exerts its function in the lumen of bile canaliculi (20A). DPPIV is homologous with CD26 (21A,22A) which has a receptor function for T cell activation and can bind to collagen (23A-26A). The CD26 molecule can become heavily glycosylated and sialylated and this regulates its receptor function strongly. For example, it is involved in several immune-mediated diseases, including AIDS. It can act as a binding protein for HIV (27A) but only when it is heavily sialylated (28A). Inhibition of DPPIV activity by specific tripeptides has an immuno suppressive effect in vivo (29A).
Amino acid synthesis and coupling reaction technology of the art includes but is not limited to U.S. Pat. Nos. 3,886,132; 4,318,905; 4,587,046; 4,771,124; 4,837,305; 5,134,232; 5,527,882, 5,602,288 and 5,637,759, all of which are incorporated herein by reference in their entirety.
All patents, references, articles, publications, standards and the like are incorporated herein by reference in their entirety.
As can be seen from the above background, a need exists to be able to determine easily the presence of an active enzyme in a cell or tissue. The present invention provides such a detection method and a kit for easy use.